Este trabalho foi financiado, em parte, por uma concessão do NIH (RO1 HL098228) para CKB.

NOTA: Todos os procedimentos com animais foram aprovados pelo Comitê Institucional Hospital Animal Care e Use do Nationwide Children. 1. Graft Manufacturing <ol><li> Tornar a solução ε-caprolactona e L-lactido copolímero P (AL / CL) por adição de 100 mg P (AL / CL) em 2 ml de dioxano sob uma coifa. Colocar a solução em um vórtice e misturar continuamente durante 1-1,5 horas para dissolver completamente. </li><li> Enquanto isso, retire a folha de ácido poliglicólico (PGA) senti do freezer e cortar várias seções 5 x 8 mm. Também cortou a ponta de uma pipeta mL 0,1-10 logo acima do filtro. </li><li> Inserir uma agulha 19 G (1,5 comprimento) na extremidade distal de uma ponta de pipeta e enrole o PGA sentiu ao redor da agulha usando micro fórceps. </li><li> Cuidadosamente empurre o feltro para a extremidade distal da ponta de pipeta, em que o lúmen é mais reto do que a parte proximal, usando uma agulha 18 G romba, enquanto a agulha G 19, é inserida no mesmo. </li><li> PipetaSolução de 40 ul de P (CL / LA) para a ponta da pipeta a partir do topo. Saturar o PGA sentida com a solução. Em seguida, empurre as bolhas de ar, utilizando um dispensador de pipeta. Repita esse processo, se necessário. </li><li> Colocar enxertos em um tubo de 50 ml e colocá-lo em um congelador a -80 ° C durante 20 min. Certifique-se que a cabeça das agulhas voltada para baixo. </li><li> Transferir o tubo para um liofilizador e vácuo durante 24 horas. Certifique-se de abrir a tampa do tubo de ter fluxo de ar. </li><li> Retire os enxertos e removê-los das agulhas. Cortar ambas as extremidades do enxerto deixando uma secção de ~ 5 mm e colocá-los de volta para as agulhas para manter a sua forma. Manter os enxertos num exsicador. </li><li> Coloque o andaime sob luz UV em um biossegurança capô O / N antes da semeadura de células. </li></ol> 2. Medula Óssea Mononuclear colheita de células e isolamento <ol><li> Eutanásia os ratos com uma sobredosagem de cetamina / xilazina (cetamina 200 mg / kg e xilazina, 20 mg / kg). </li><li> Retire os ossos (fêmur umd tíbias) a partir de 3 camundongos por 10 implantações de enxerto e coloque em uma placa de Petri com 10 cc RPMI. Corte ambas as extremidades dos ossos e da medula óssea nivelada, utilizando uma seringa com uma agulha de 25 G em uma nova placa de Petri com 3 cc RPMI. Recolha de medula óssea e solução de RPMI em um tubo de 15 cc e lava-se a placa de Petri com meio RPMI de 2 cc adicionais para recolher o remanescente da BM. </li><li> Tome amostra (5-10 mL) e contagem de células utilizando um contador de células automatizado ou hemocitômetro. Anote o resultado. </li><li> Colocar 5 ml de Ficoll em um tubo de centrífuga de 15 cc e adicionar medula óssea e solução de RPMI. Adicionar a solução muito cuidadosamente para evitar que a mistura com Ficoll. </li><li> Centrifugar a 528 xg por 30 minutos com &quot;NO FREIO&quot; a 24 ° C. </li><li> Retire a camada rosa superior. Recolhe-se o meio clara camada Figura 1, que é a camada de EMN, e diluir com PBS 01:01. </li><li> Centrifugar a solução diluída de MNC a 528 xg durante 10 min a 24 ° C. </li><li> Remover o sobrenadante e dilute a pelota com 5 ml de PBS. </li><li> Centrifugar a solução do agregado a 528 xg durante 10 min a 24 ° C. </li><li> Remover o sobrenadante. Diluir o sedimento com o volume apropriado de meio RPMI (~ 200 mL). </li><li> Tomar uma amostra mL 5-10 e contagem de células utilizando um contador de células automatizado ou hemocitômetro. Anote o resultado. Repetir a contagem de células mais uma vez e calcular o número médio de célula. </li><li> Dilui-se a concentração das células para um milhão células/10 ul usando RPMI. </li></ol> 3. Sementeira celular <ol><li> Andaime Prewet pela adição de 5 mL RPMI luminally por 5 min, em seguida, retire RPMI. </li><li> Adicionar 10 mL de medula óssea derivados mono células nucleares em RPMI a partir do Passo 2,12 a lúmen andaime e aguarde 10 minutos para permitir que as células para anexar para o cadafalso. </li><li> Cortar uma agulha 19 G 1 cm de comprimento e colocar a agulha para dentro do lúmen do andaime para manter a forma do andaime. Coloque a amostra em uma placa de 24 poços. &lt;/ Li&gt; <li> Adicionar 1000 mL de RPMI a cada poço e incubar S / N na incubadora. </li></ol> 4. Graft Implantation <ol><li> Autoclave todos os instrumentos cirúrgicos antes da cirurgia: 1x tesoura fina, micro fórceps 3x, pinças vasculares micro 2x, braçadeira 1x fórceps de aplicação, suporte da agulha micro 1x, tesoura primavera 1x, 1x afastador. </li><li> 6-8 semanas de idade do sexo feminino C57BL / 6 são usados ​​como engenharia de tecidos receptores de enxertos vasculares. Remover o rato da sua gaiola e pesá-lo, em seguida, através de anestesiar uma injecção intraperitoneal no quadrante inferior direito do abdómen com um cocktail de cetamina / xilazina (cetamina 100 mg / kg e xilazina, 10 mg / kg). O cetoprofeno (5 mg / kg, IP) é usado como um analgésico de pré-anestesia. </li><li> Verifique o nível de sedação por beliscar conto, em seguida, prenda o cabelo abdominal. Lubrifique os olhos com pomada oftálmica estéril, e coloque o mouse em uma posição de decúbito dorsal sobre uma almofada. Desinfetar o abdômen com Betadine e álcool pads. CoVer o mouse com um campo estéril e expor apenas a área da incisão. </li><li> Faça uma laparotomia mediana incisão abaixo do xifóide até a região suprapúbica, e inserir um afastador auto-retenção. Enrole os intestinos em salina umedecido gaze. Sem rodeios definir a aorta infra-renal e veia cava. </li><li> Coloque duas pinças vasculares micro em ambos os lados proximal e distal da aorta ea veia cava, em seguida, sem rodeios separar a aorta da veia cava Transecto veia cava. Se necessário, ligadura dos ramos da aorta abdominal com 10-0 suturas monofilamento as agulhas cônicos. </li><li> Implantar uma veia cava inferior, com interposição de enxerto extremidade proximal e distal à anastomose de acabar usando uma estéril 10-0 sutura. Apare o enxerto, geralmente 1-2 mm, dependendo da anatomia do mouse. Fixar o enxerto com um ponto em ambas extremidades proximais e distais e iniciar a sutura contínua com 4-5 stiches do outro lado do enxerto. Depois de terminar a parte da frente, vire as braçadeiras e enxertospara o outro lado e suturar o lado de trás do enxerto. Durante a implantação, lave o enxerto com solução de heparina com frequência para prevenir a trombose aguda. </li><li> Retirar a braçadeira proximal e controlar a hemorragia através da aplicação de um agente hemostático estéril absorvível tópica. Quando a hemorragia parar completamente, retirar a pinça distai e controlar a hemorragia da mesma maneira. Faça fluxos sanguíneos certeza através do enxerto. </li><li> Feche a musculatura abdominal e pele em duas camadas usando uma sutura de monofilamento 6-0 poliamida preto com agulha rosqueada incluído. </li><li> Injetar 0,5 ml por via subcutânea de solução salina e coloque o rato em uma gaiola de recuperação em um bloco de aquecimento até que o mouse é totalmente móvel. Após a recuperação, o retorno do mouse para uma nova gaiola com roupa de cama de papel. Dar medicação para dor (Ibuprofeno, 30 mg / kg, a água potável) para 48 horas. </li></ol>

<ol>
	<li>Heart Association, A. m. e. r. i. c. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics&#8212;2012+Update.">Heart Disease and Stroke Statistics&#8212;2012 Update.</a> Circulation. 125, (2012).</li><li>Shinoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+Of+Viable+Pulmonary+Artery+Autografts+Through+Tissue+Engineering.">Creation Of Viable Pulmonary Artery Autografts Through Tissue Engineering.</a> The Journal of Thoracic and Cardiovascular Surgery. 115, 536-546 (1998).</li><li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late-term+results+of+tissue-engineered+vascular+grafts+in+humans.">Late-term results of tissue-engineered vascular grafts in humans.</a> The Journal of Thoracic and Cardiovascular Surgery. 139, 431-436 (2010).</li><li>Shin'oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midterm+clinical+result+of+tissue-engineered+vascular+autografts+seeded+with+autologous+bone+marrow+cells.">Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 129, 1330-1338 (2005).</li><li>Brennan, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+vascular+grafts+demonstrate+evidence+of+growth+and+development+when+implanted+in+a+juvenile+animal+model.">Tissue-engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model.</a> Ann Surg. 248, 370-377 (2008).</li><li>Roh, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+autologous+tissue-engineered+venous+conduit+from+bone+marrow-derived+vascular+cells:+optimization+of+cell+harvest+and+seeding+techniques.">Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques.</a> Journal of Pediatric Surgery. 42, 198-202 (2007).</li><li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+vascular+grafts+form+neovessels+that+arise+from+regeneration+of+the+adjacent+blood+vessel.">Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel.</a> The FASEB Journal. 25, 2731-2739 (2011).</li><li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+macrophages+in+neovessel+formation+and+the+development+of+stenosis+in+tissue-engineered+vascular+grafts.">A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts.</a> The FASEB Journal. 25, 4253-4263 (2011).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Natural+History+of+Extracellular+Matrix+Production+in+Tissue-Engineered+Vascular+Grafts+during+Neovessel+Formation.">Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation.</a> Cells Tissues Organs. 195, 60-72 (2012).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Burst+Pressure:+Initial+Evaluation+of+the+Natural+History+of+the+Biaxial+Mechanical+Properties+of+Tissue+Engineered+Vascular+Grafts+in+the+Venous+Circulation+Using+a+Murine+Model.">Beyond Burst Pressure: Initial Evaluation of the Natural History of the Biaxial Mechanical Properties of Tissue Engineered Vascular Grafts in the Venous Circulation Using a Murine Model.</a> Tissue Eng. Part A. 20, (2013).</li><li>Mirensky, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+vascular+grafts:+does+cell+seeding+matter.">Tissue-engineered vascular grafts: does cell seeding matter.</a> Journal of Pediatric Surgery. 45, 1299-1305 (2010).</li><li>Roh, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+vascular+grafts+transform+into+mature+blood+vessels+via+an+inflammation-mediated+process+of+vascular+remodeling.">Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.</a> Proceedings of the National Academy of Sciences. 107, 4669-4674 (2010).</li><li>Mirensky, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+arterial+grafts:+long-term+results+after+implantation+in+a+small+animal+model.">Tissue-engineered arterial grafts: long-term results after implantation in a small animal model.</a> Journal of Pediatric Surgery. 44, 1127-1133 (2009).</li><li>Lee, Y. U., Naito, Y., Kurobe, H., Breuer, C. K., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biaxial+mechanical+properties+of+the+inferior+vena+cava+in+C57BL/6+and+CB-17+SCID/bg+mice.">Biaxial mechanical properties of the inferior vena cava in C57BL/6 and CB-17 SCID/bg mice.</a> Journal of Biomechanics. 46, 2277-2282 (2013).</li></ol>

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O modelo de rato de TEVG é uma ferramenta valiosa para estudar os mecanismos celulares e moleculares da formação neotissue e o desenvolvimento de estenose. O semeado BM-MNC foi mostrado em ambas as imagens histológicas e SEM das células semeadas sobre o enxerto 11. Eficiência sementeira celular também foi demonstrado utilizando um ensaio de ADN 7. Utilizando este sistema modelo, mostrou que a sementeira de células, reduz a incidência do desenvolvimento de estenose TEVG, que foi o modo prin...

As cardiopatias congênitas são doenças graves que afetam cerca de 8% dos nascidos vivos nos Estados Unidos. Aproximadamente 25% desses recém-nascidos com defeitos cardíacos congênitos ou 2,4 por 1.000 nascidos vivos, necessitam de tratamento invasivo no primeiro ano de sua vida 1. O tratamento mais eficaz para a doença cardíaca congênita é a cirurgia reconstrutiva. Infelizmente, as complicações decorrentes da utilização de condutas vasculares actualmente disponíveis são a causa mais importante de morbidade e mortalidade no pós-operatório. Para resolver este problema, desenvolvemos os primeiros da engenharia de tecidos enxertos vasculares (TEVGs) para uso clínico 2. TEVGs foram construídos a partir de tubos de poliéster biodegradável semeados com medula óssea autólogo de células derivadas de mononucleares (BM-PTM) e implantados como canais venosos para cirurgia cardíaca congênita. Os resultados mostraram excelentes taxas de permeabilidade em 1-3 anos de follow-up, mas com significativa incidência de estenose <saté&gt; 3,4. Ficou claro que era necessária uma melhor compreensão da formação neotissue vascular eo mecanismo subjacente ao desenvolvimento de estenose TEVG. A fim de compreender melhor o desenvolvimento de TEVGs e o mecanismo do desenvolvimento de estenose, um modelo de ovino foi criado 5,6. Neste modelo, os TEVGs transformado com sucesso em vasos de vida e foram semelhantes em ambos morfologia e função das veias nativas. Este uso de um modelo animal de grande porte foi um bom primeiro passo na prestação de informações pré-clínica importante que ajudou o uso clínico de TEVGs. No entanto, a plena compreensão dos mecanismos celulares e moleculares da formação neotissue vascular em TEVGs usando grandes modelos animais é limitada devido às limitações na caracterização molecular de fenótipos celulares vasculares devido à falta de espécies ferramentas moleculares específicos. Para superar essas limitações, um modelo murino de TEVGs foi desenvolvido em razão do rápido avanço na genética do rato e sua extensa moleculaCaracterização r, com a vantagem adicional de uma escala de tempo encurtado. O modelo murino de interposição IVC fielmente recapitulado o processo de formação Neovaso formado que ocorre em animais de grande porte e seres humanos, mas ao longo de um curso de tempo muito mais curto de 6-9. Aqui, um protocolo detalhado para a fabricação de enxerto de pequena escala utilizando matrizes biodegradáveis, foram descritos colheita BM-MNC e isolamento, BM-MNC semeadura no andaime, e implantação de enxerto no modelo murino.

<table border="0" cellpadding="0" cellspacing="0" style="width:616px;" width="615">
	<colgroup>
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		<col />
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Name</td>
			<td style="width:139px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:168px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polyglycolic acid (PGA) felt</td>
			<td>Biomedical Structures</td>
			<td></td>
			<td>Custome ordered</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:176px;">&#603;-caprolactone and L- 
			lactide copolymer P(LA/CL)&#160;</td>
			<td>Gunze Inc.</td>
			<td></td>
			<td>Custome ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipet tip, 0.1-10 &#956;l&#160;</td>
			<td>Fisher Sientific</td>
			<td>02-707-456</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lyophilizer&#160;</td>
			<td>Labconco</td>
			<td>7070020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI medium 1604</td>
			<td>gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Petri dish</td>
			<td>BD</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:176px;">24 well plate</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15cc tube&#160;</td>
			<td>BD</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Ficoll</td>
			<td>Sigma</td>
			<td>10831-100ml</td>
			<td>Also called &#39;Histopaque&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">DPBS</td>
			<td>gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Littauer Bone Cutter 4.5&#34; Straight</td>
			<td>Roboz</td>
			<td>RS-8480</td>
			<td>For BM harvesting</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:176px;">Forceps 4.5&#34;</td>
			<td>Roboz</td>
			<td>RS-8120</td>
			<td>For BM harvesting</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Scissors 4.5&#34;</td>
			<td>Roboz</td>
			<td>RS-5912</td>
			<td>For BM harvesting</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:176px;">Microscope</td>
			<td style="width:139px;">Leica</td>
			<td style="width:133px;">M80</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:176px;">C57BL/6J (H-2b), Female</td>
			<td style="width:139px;">Jackson Laboratories&#160;</td>
			<td style="width:133px;">664</td>
			<td style="width:168px;">8-12 weeks</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Ketamine Hydrochloride Injection</td>
			<td style="width:139px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-2053</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Xylazine Sterile Solution</td>
			<td style="width:139px;">Akorn Inc.</td>
			<td style="width:133px;">NADA# 139-236</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">ketoprofen</td>
			<td style="width:139px;">Fort Dodge Animal Health</td>
			<td style="width:133px;">NDC 0856-4396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">Ibuprofen</td>
			<td style="width:139px;">PrecisionDose</td>
			<td style="width:133px;">NDC 68094-494-59</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Heparin Sodium</td>
			<td style="width:139px;">Sagent Pharmaceticals</td>
			<td style="width:133px;">NDC 25021-400</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Saline solution (Sterile 0.9% Sodium Chloride)</td>
			<td style="width:139px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-0138-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">0.9% Sodium Chloride Injection</td>
			<td style="width:139px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-4888-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:176px;">Petrolatum Ophthalmic Ointment</td>
			<td style="width:139px;">Dechra Veterinary Products</td>
			<td style="width:133px;">NDC 17033-211-38</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Iodine Prep Pads</td>
			<td style="width:139px;">Triad Disposables, Inc.</td>
			<td style="width:133px;">NDC 50730-3201-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Alcohol Prep Pads</td>
			<td style="width:139px;">McKesson Corp.</td>
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			<td height="23" style="height:23px;width:176px;">Micro-Adson Forcep</td>
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			<td height="20" style="height:20px;width:176px;">Dumont #5 Forcep</td>
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			<td height="38" style="height:38px;width:176px;">Black Polyamide Monofilament Suture, 10-0</td>
			<td style="width:139px;">AROSurgical Instruments Corporation</td>
			<td style="width:133px;">TI638402</td>
			<td style="width:168px;">For sutureing the graft</td>
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		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Black Polyamide Monofilament Suture, 6-0</td>
			<td style="width:139px;">AROSurgical Instruments&#160;</td>
			<td style="width:133px;">SN-1956</td>
			<td style="width:168px;">For musculature and skin closure</td>
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			<td height="20" style="height:20px;width:176px;">Non-Woven Songes</td>
			<td style="width:139px;">McKesson Corp.</td>
			<td style="width:133px;">94442000</td>
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			<td height="24" style="height:24px;width:176px;">1 ml Syringe</td>
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			<td height="24" style="height:24px;width:176px;">10 ml Syringe</td>
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			<td height="20" style="height:20px;width:176px;">18G 1 1/2 in, Needle</td>
			<td style="width:139px;">BD</td>
			<td style="width:133px;">305190</td>
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			<td height="20" style="height:20px;width:176px;">25G 1 in., Needle</td>
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			<td height="20" style="height:20px;width:176px;">30G 1 in., Needle</td>
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			<td height="23" style="height:23px;width:176px;">Warm Water Recircultor</td>
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			<td style="width:133px;">TP-700</td>
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			<td height="23" style="height:23px;width:176px;">Warming Pad</td>
			<td style="width:139px;">Gaymar</td>
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			<td height="20" style="height:20px;width:176px;">Trimmer</td>
			<td style="width:139px;">Wahl</td>
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implantation of inferior vena cava interposition graft in mouse model

Scaffolds biodegradáveis ​​semeado com células mononucleares da medula óssea (BMCS) são muitas vezes utilizados para a cirurgia reconstrutiva para tratar anomalias cardíacas congênitas. Os resultados clínicos a longo prazo mostrou excelentes taxas de permeabilidade, porém, com significativa incidência de estenose. Para investigar os mecanismos celulares e moleculares da formação neotissue vascular e prevenir o desenvolvimento de estenose em engenharia de tecidos enxertos vasculares (TEVGs), foi desenvolvido um modelo do rato do enxerto com aproximadamente 1 mm de diâmetro interno. Em primeiro lugar, os TEVGs foram montados a partir de andaimes tubulares biodegradáveis ​​fabricadas a partir de um tecido não urdido de ácido poliglicólico sentida malha revestida com ε-caprolactona e L-lactido copolímero. Os suportes foram então colocados num liofilizador, aspirado durante 24 h, e armazenado num excicador até sementeira celular. Em segundo lugar, a medula óssea foi colhida a partir de ratinhos dadores e as células mononucleares foram isoladas por centrifugação em gradiente de densidade. Em terceiro lugar, aproximadamente um milhão de células foramsemeadas num andaime e incubadas O / N. Finalmente, os andaimes sem sementes foram então implantados como infra-enxertos na veia cava de interposição em camundongos C57BL / 6. Os enxertos implantados demonstraram excelente patência (&gt; 90%) sem evidência de complicações tromboembólicas ou formação de aneurisma. Este modelo murino nos ajudará a compreender e quantificar os mecanismos celulares e moleculares da formação neotissue no TEVG.

Um esquema de implantação TEVG é mostrado na Figura 1. Medula óssea foi colhida a partir de um rato dador e células mono nucleares foram isolados usando centrifugação de densidade e em seguida semeadas num andaime biodegradável. Os andaimes semeados foram incubados O / N e implantado a um mouse destinatário como uma veia cava inferior interposição de enxerto. A figura 2 ilustra a microscopia eletrônica de varredura da PGA-P (CL / LA) andaime. O d...

Watch this Scientific Journal Video about Implantation of Inferior Vena Cava Interposition Graft in Mouse Model at JoVE.com

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Para melhorar o nosso conhecimento da formação neotissue celular e molecular, um modelo murino da TEVG foi recentemente desenvolvida. Os enxertos foram implantados como infra-enxertos na veia cava de interposição em camundongos C57BL / 6. Este modelo consegue resultados semelhantes aos obtidos em nossa investigação clínica, mas ao longo de um curso de tempo muito reduzido.

Implantação de Veia Cava Inferior interposição de enxerto no Rato Modelo

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

implantation-of-inferior-vena-cava-interposition-graft-in-mouse-model

Research

JoVE Journal

Medicine

11.8K visualizações.  Nationwide Children's Hospital.  Para melhorar o nosso conhecimento da formação neotissue celular e molecular, um modelo murino da TEVG foi recentemente desenvolvida. Os enxertos foram implantados como infra-enxertos na veia cava de interposição em camundongos C57BL / 6. Este modelo consegue resultados semelhantes aos obtidos em nossa investigação clínica, mas ao longo de um curso de tempo muito reduzido. 

Vídeo: Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential therapeutic compounds (1). New compounds to be utilized in the treatment and prevention of DVT are currently being developed. The delivery of potential therapeutic antagonist compounds to an affected thrombosed vein has been problematic. In the context of therapeutic applications, a model that uses partial stasis and consistently generates thrombi within a major vein has been recently established. The Electrolytic Inferior vena cava Model (EIM) is mouse model of DVT that permits thrombus formation in the presence of continuous blood flow. This model allows therapeutic agents to be in contact with the thrombus in a dynamic fashion, and is more sensitive than other models of DVT (1). In addition, this thrombosis model closely simulates clinical situations of thrombus formation and is ideal to study venous endothelial cell activation, leukocyte migration, venous thrombogenesis, and to test therapeutic applications (1). The EIM model is technically simple, easily reproducible, creates consistent thrombi sizes and allows for a large sample (i.e. thrombus and vein wall) which is required for analytical purposes.

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

The electrolytic induction of endothelial activation to the internal surface of the Inferior Vena Cava results in venous type thrombus formation due to endothelial activation and partial blood stasis, two components of Virchow's triad.

Eletrolítico Veia Cava Inferior Model (EIM) de trombose venosa

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Medicina

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

Modelo do mouse completa de Stasis Inferior Trombose da veia cava

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Treinar um técnica microcirúrgica sofisticado: Interposição de enxerto de veia jugular externa na artéria carótida comum em ratos

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Veia Interposição Modelo: Um modelo adequado para estudar Bypass Graft Perviedade

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

Remendar Angioplastia na Rat aorta ou veia cava inferior

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

Um modelo do rato de ortotópico transplante hepático, usando uma técnica de anastomose magnético romance para reconstrução de veia Cava Suprahepatic

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Rodent Model of The Ross Operation: Syngeneic Pulmonary Artery Graft Implantation in A Systemic Position

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding root replacement, the possible dilation of the pulmonary autograft and subsequent aortic regurgitation is described. Several animal models have been proposed. However, these are usually limited to ex-vivo models or in-vivo experiments with relatively expensive large animal models. In this study, we sought to establish a rodent model of pulmonary artery graft (PAG) implantation in a systemic position. A total of 39 adult Lewis rats were included. Immediately after euthanasia, the pulmonary root was harvested from a donor animal (n=17). Syngeneic recipient (n=17) and sham-operated (n=5) rats were sedated and ventilated. In the recipient group, the PAG was implanted with an end-to-end anastomosis in infra-renal abdominal aortic position. Sham-operated rats underwent only transection and re-anastomosis of the aorta. Animals were followed with serial ultrasound studies for two months and post-mortem histological analysis. The median PAG diameter in the native position was 3.20 mm (IQR=3.18-3.23). At follow-up, the median diameter of the PAG was 4.03 mm (IQR=3.74-4.13) at 1 week, 4.07 mm (IQR=3.80-4.28) at 1 month, and 4.27 mm (IQR=3.90-4.35) at 2 months (p&lt;0.01). Peak systolic velocity was 220.07 mm/s (IQR=210.43-246.41) at 1 week, 430.88 mm/s (IQR=375.28-495.56) at 1 month, and 373.68 mm/s (IQR=305.78-429.81) at 2 months (p=0.02) and did not differ from the sham-operated group at the end of the experiment (p=0.5). Histological analysis did not show any sign of endothelial thrombosis. This study showed that rodent models may allow for the evaluation of the long-term adaptation of the pulmonary root to a high-pressure system. A systemically placed syngeneic PAG implantation represents a simple and feasible platform for the development and evaluation of novel surgical techniques and drug therapies to further improve the outcomes of the Ross operation.

We demonstrate how to establish a murine model of pulmonary root implantation into the descending aorta to simulate the Ross procedure. This model enables the medium/long-term evaluation of pulmonary autograft remodeling in a systemic position, representing the basis of developing therapeutic strategies to promote its adaptation.

Um modelo de roedor da operação Ross: Implantação de enxerto de artéria pulmonar sinténica em posição sistêmica

The Ross operation for aortic valve disease has regained new interest due to its outstanding long-term results. Nonetheless, when employed as freestanding ...

Image Acquisition Method for the Sonographic Assessment of the Inferior Vena Cava

Over the past several decades, clinicians have incorporated several applications of diagnostic point-of-care ultrasound (POCUS) into medical decision-making. Among the applications of POCUS, imaging the inferior vena cava (IVC) is practiced by a wide variety of specialties, such as nephrology, emergency medicine, internal medicine, critical care, anesthesiology, pulmonology, and cardiology. Although each specialty uses IVC data in slightly different ways, most medical specialties, at minimum, attempt to use IVC data to make predictions about intravascular volume status. While the relationship between IVC sonographic data and intravascular volume status is complex and highly context-dependent, all clinicians should collect the sonographic data in standardized ways to ensure repeatability. This paper describes standardized IVC image acquisition including patient positioning, transducer selection, probe placement, image optimization, and the pitfalls and limitations of IVC sonographic imaging.&#160;This paper also describes the commonly performed anterior IVC long-axis view and three other views of the IVC that can each provide helpful diagnostic information when the anterior long-axis view is difficult to obtain or interpret.

Point-of-care ultrasound evaluation of the inferior vena cava (IVC) is commonly utilized to identify, among other things, the volume status. Imaging should be performed systematically to ensure repeatability. This manuscript reviews the methods and pitfalls of sonographic IVC examination.

Método de aquisição de imagens para avaliação ultrassonográfica da veia cava inferior

Over the past several decades, clinicians have incorporated several applications of diagnostic point-of-care ultrasound (POCUS) into medical ...

Estratégias para Anastomose Vascular Eficiente em Transplante de Fígado de Camundongo

Vascular Reconstruction with the Cuff Technique in Mouse Orthotopic Liver Transplantation

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. However, the technical challenges pose a barrier to utilizing this valuable experimental model and passing on these skills to the next generation. The most challenging aspect of this procedure is vascular reconstruction, including the portal vein (PV), infrahepatic inferior vena cava (IHIVC), and suprahepatic inferior vena cava. The use of plastic cuffs, rather than sutures, allows for smoother PV and IHIVC reconstruction. Vessels are reconstructed by attaching a cuff made from an intravenous catheter to the tip of the graft vessel and interposing the cuff into the recipient vessel. The two most crucial aspects are properly visualizing the inner lumen of the vessel and avoiding the use of excessive force. Our aim is to provide a technical overview of vascular reconstructions using the cuff technique in recipient surgery. These technical tips for the cuff technique are expected to help microsurgeons facilitate vascular reconstruction and advance their research.

Autor em destaque: Explorando a isquemia fria e a lesão de reperfusão quente no transplante de fígado gorduroso

This protocol provides technical information for vessel reconstruction using the cuff technique in mouse orthotopic liver transplantation.

Reconstrução Vascular com a Técnica do Manguito no Transplante Hepático Ortotópico de Camundongo

Mouse orthotopic liver transplantation is an effective methodology for investigating the underlying mechanisms of liver ischemia and reperfusion injury. ...